Method of determining the sequence of nucleic acids employing solid-phase particles carrying transponders

ABSTRACT

A method is described for determining the sequence of nucleic acids. The method employs small solid phase particles having transponders, with a primary layer of an oligonucleotide of known sequence attached to the outer surface of the particle. A read/write scanner device is used to encode and decode data on the transponder. The stored data includes the sequence of the oligonucleotide immobilized on the transponder. The sequence of sample nucleic acids is determined by detecting annealing to an oligonucleotide bound to a particle, followed by decoding the transponder to determine the sequence of the oligonucleotide.

This application is a divisional of application Ser. No. 08/564,860,filed Nov. 30, 1995, now U.S. Pat. No. 5,736,332.

BACKGROUND OF THE INVENTION

This invention relates to materials and methods for determining thesequence of unknown or target nucleic acids, and more specifically tomaterials and methods for determining the sequence of target nucleicacids using an electronically-indexed solid phase, with transpondersassociated with the solid phase particles.

A high throughput method for ascertaining the sequence of sample nucleicacids is sequencing by hybridization (SBH). In that method, a largenumber of oligonucleotide probes is allowed to interact with the nucleicacid molecules in a sample, and a detection system is provided todetermine whether individual oligonucleotides have annealed to thetemplate. Two basic designs have been described. In one, theoligonucleotide probes are arranged in a two-dimensional array on thesurface of a membrane, filter, VLSI chip, or the like. In the other, thearray on the membrane is formed with a large number of sample DNAsequences, and each membrane is subjected to a series of hybridizationsteps with different oligonucleotide probes. A label used to monitor thebinding, either a radioactive isotope or a fluorophore, is carried onthe sample DNA or on the oligonucleotide probe. The sequence is derivedfrom coordinates of spots showing a high level of the label depositionon the two-dimensional arrays.

Conventional SBH methods are limited by difficulties related topreparation of arrays of DNA, non-specific annealing of DNAs, the needfor special instrumentation to read the data and the automation of theprocess and data analysis. In SBH, the sequence is determined bydefining the two-dimensional coordinates of relevant dots in the arrayformed by the deposited DNA molecules. In the present invention, apartial or the complete sequence of the DNA molecule is determined bydecoding the electronic memory elements associated with DNA probes ofknown sequence.

An advantage of the present invention over conventional sequencingmethods is that it is extremely fast, because the sequence is deducedfrom a series of readings of digitally-stored sequences in thetransponder, rather than from a series of measurements of a chemical orphysical property of DNA, or the location of DNA in an array. The methodof this invention is referred to hereinafter as digital sequencing.

SUMMARY OF THE INVENTION

The present invention overcomes the problems of conventional sequencingmethods by employing solid phase particles having a transponderassociated with each particle. The particles carry oligonucleotideprobes attached to their surface, and the sequence of theoligonucleotide is encoded on a memory element on the transponder. Theoligonucleotide probes correspond to subsequences believed to exist inthe target sequence.

To determine the sequence of sample, or "target" DNA, the target DNA ofunknown sequence is labeled with a fluorophore and combined withtransponder particles carrying known oligonucleotides under annealingconditions. The transponders are analyzed to detect the fluorescence orcolor originating from a label that indicates that target DNA has boundto the probe attached to the surface of the transponder, and theinformation stored electronically in the transponder is decoded.Dedicated sequence analysis software may then be used to determine thecomplete or partial sequence of the DNA target.

In one aspect, the present invention provides a solid phase particle foruse in determining the sequence of nucleic acids, comprising a solidphase particle having a transponder, and an oligonucleotide probe havinga known sequence attached to an outer surface of the particle.

In another aspect, the present invention provides a method ofdetermining the sequence of sample nucleic acids, comprising the stepsof employing solid phase particles with transponders.

In another aspect, the invention provides a kit for determining thesequence of unknown nucleic acids, comprising an assay vessel, and a setof solid phase particles having transponders, and a differentoligonucleotide attached to the surface of the probe.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a sequencing procedure of thisinvention.

FIG. 2 is a cross-sectional view of a solid phase particle with atransponder, and a primary layer of biomolecules bound to a surfacethereof.

FIG. 3 is a schematic diagram of the signal pathway for encoding anddecoding data on the transponders of the solid phase.

FIG. 4 is a schematic representation of a miniature transponder.

FIG. 5 is a plan view of a miniature transponder.

FIG. 6 is a plan view of a transport system/analytical instrument forimplementing the present invention.

FIG. 7 is a plan view of a modified flow cytometer for high speedanalysis of solid phase particles of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 depicts a simple sequencing procedure of the current invention. Asolid phase particle 10 with a transponder 12 is derivatized byattaching an oligonucleotide probe 14 of known sequence to the outersurface 16 of the particle 10. The transponder 12 is encoded with anindex number that indicates the sequence of the oligonucleotide probe14. The particle 10 is immersed in a solution containing labelled targetDNA 11, and the solution is heated to cause DNA to dissociate, thencooled to allow the DNA to anneal, resulting in target DNA 11 annealingto the probe 14.

The transponder 12 is analyzed to detect the fluorescence of any targetDNA 11 bound to the transponder 12, and the transponder 12 is decodedusing a read/write scanner device (not shown). In practice a group oftransponders, each carrying a different probe and each encoded with thesequence of that probe would be used.

The target DNA is preferably pre-treated by digestion with anappropriate restriction endonuclease, or fragmented by digesting withDNase I, to yield relatively short (preferably 10 nt to 100 nt)preferably single-stranded DNA. Pretreatment may also involve aconversion of DNA to RNA by cloning and in vitro transcription, followedby a partial hydrolysis of RNA (if necessary), or a generation of a DNAfragment by PCR, and the latter can be coupled with labeling of DNA witha fluorophore. DNA provided for sequencing may already be in thepreferred form. If, however, the DNA is in the form of a longdouble-stranded DNA molecule, a long single-stranded DNA molecule, aclosed circular DNA molecule, or a nicked circular double-stranded DNAmolecule, the DNA must be pre-treated.

Labelling sample DNA with a fluorogenic hapten before annealing is apractical, although not always necessary, step in digital sequencing.Restriction fragments with the 5'-protruding end can be labeled in achain extension reaction with a DNA polymerase utilizing one or more ofdNTPs derivatized with a fluorogenic hapten. 3'-ends of DNA moleculescan be labeled in an enzymatic reaction employing a terminaldeoxynucleotidyl transferase (TdT) and dNTP derivatized with afluorogenic hapten. In some sandwich-type applications, the fluorogenichapten can be replaced with any hapten (e.g. biotin) for which anantibody (or a binding partner) is available, the goal being to detectthe binding of labeled DNA to the transponders through a sandwichconfiguration involving an anti-hapten antibody and a secondaryanti-antibody antibody conjugated to an enzyme, said enzyme catalyzing areaction with a precipitating fluorogenic substrate.

Several types of nucleic acid can be immobilized on the transponders.They include DNA, RNA and modifications thereof, such as protein-nucleicacid (PNA) molecules. It is preferred that the immobilized nucleic acidsare single-stranded. The length of the immobilized nucleic acids canvary in different implementations of the digital sequencing, therequirement being that the length should be sufficient to provide adesired level of binding specificity to the target DNA. In oneembodiment of this invention, the oligonucleotide probes correspond tosubsequences that are believed to exist in the target nucleic acidsequence.

There are several methods to immobilize nucleic acids on the solid phaseparticles of this invention, including the conjugation ofoligonucleotides to the transponders, or the direct chemical synthesisof the oligonucleotide on the transponders. Combinatorial synthesis is apreferred method of direct chemical synthesis, and involves up to fourindependent condensations of a four different nucleosides (A, C, G andT) on a large number of transponders in four vessels. The transpondersare divided into four pools, and each pool is reacted with a differentnucleoside. After each condensation, the transponders in the four poolsare encoded with a symbol indicating the nucleoside used in thecondensation. The four transponder pools are then combined andredistributed into four vessels, and the process is repeated as manytimes as necessary and practical. The net result of combinatorialsynthesis on transponders is that the transponders are derivatized withdifferent oligonucleotides, and the sequence of the oligonucleotide isencoded in the transponder.

The derivatized transponders and the target DNA are kept in a singlevessel in an appropriate buffer. The volume of the buffer must besufficient to completely immerse the transponders in the buffer. Anappropriate buffer for the annealing reaction is phosphate-bufferedsaline (PBS), but many other buffers are suitable, as is well-known bythose of ordinary skill in this art. The vessel is heated to atemperature typically in the range of 60°-100° C. The temperature shouldbe sufficient to allow for melting of double-stranded DNA that mightexist in the vessel into the single-stranded form. The vessel is thenslowly cooled to a temperature below the melting temperature for thesequences immobilized on the transponder, which typically is in therange of 0°-60° C., and is often room temperature. The transponders arethen washed thoroughly several times to remove unbound target DNA.

If the target DNA was fluorescence-labeled before annealing, nopost-annealing treatment is needed, and the transponders can besubjected to decoding, and the fluorescence of the surface can bemeasured. Alternatively, if the target DNA was not labeled,fluorescence-labeling must be done after annealing. Either target DNA(associated with the oligonucleotide bound to the transponder) can belabeled, or the oligonucleotide probe on the transponder (but only thosethat are associated with the target DNA) can be labeled. A methodsuitable for both approaches is chain extension utilizing a DNApolymerase.

Chain extension labeling procedures can differ with regard to: (a) thetype of the fluorophore-tagged nucleotide used for the labeling, or (b)the number of different fluorescent nucleotides used in the extensionreaction (choice of the adenine, cytosine, guanine or thyminederivatives). As to the type of nucleotide used, the nucleotide can beeither of the deoxy type, or the dideoxy type. More than one labeleddeoxy nucleotide can be incorporated into the extended portion of thechain, but the incorporation of only one dideoxy nucleotide isattainable, since it prevents further chain extension.

The labeling can be combined with a fluorescence detection method fortransponders which allows for distinguishing the type of thefluorescence label used by identifying the maximum emission wavelengthof the fluorophore, similar to the implementation in automatedsequencing on Applied Biosystems sequencers. The use of dideoxynucleotides in digital sequencing, therefore, offers the ability toidentify the residue immediately proximal to the 3' end of either targetDNA, or the oligonucleotide on the transponder, depending on theapproach used.

In a preferred embodiment of the labeling procedure in the presence offour fluorophore-labeled dideoxy nucleotide triphosphates(ddATP,ddCTP,ddGTP and ddTTP) the primers attached to the transponderthat annealed to the target are extended by one nucleotide residue by aDNA polymerase. The type of the incorporated residue (A, C, G or T) isdetermined by the sequence of the target. Therefore, the wavelength atwhich the maximum intensity of fluorescence is observed indicates theresidue type. After the extension step, the particles are passed througha fluorometer capable of discriminating between four wavelengths ofemitted light. In this approach, more information is obtained than inthe basic implementation of the method. As previously, the presence ofthe fluorescence is an indication that the annealing took place, but nowthe wavelength of the fluorescence additionally identifies the targetresidue that is immediately downstream from the primer (i.e. close tothe 3' end of the primer).

In an alternative version of the above procedure, only one fluorophoreis used in the reaction, but four separate extension reactions areperformed in four separate vessels employing one of four ddNTPs in eachvessel. After the reaction and appropriate washes, the particles fromthe four vessels are mixed together, and the remainder of the procedureis carried through as presented above. In this case, however, anadditional encoding step of the particle's memory is needed to providethe information about the type of nucleotide used in the extensionreaction for the given transponder.

FIG. 2 depicts a solid phase particle for use in this invention. Theparticle 10 is derivatized by attaching an oligonucleotide probe 14 ofknown sequence to a surface 16 of the particle 10. A transponder 12 isassociated with the particle 10. The transponder 12 is encoded with anindex number that indicates the sequence of the probe 14. Thetransponder may be pre-programmed by the manufacturer, or it may beencoded by the user, using a scanner read/write device.

A transponder is a radio transmitter-receiver activated for transmissionof data by reception of a predetermined signal, and may also be referredto as a microtransponder, radiotransponder, radio tag, transceiver, etc.The signal comes from a dedicated scanner, which also receives andprocesses the data sent by the transponder in response to the signal.The scanner function can be combined with the write function, i.e. theprocess of encoding the data on the transponder. Such a combinationinstrument is called a scanner read/write device. An advantage of thetransponder-scanner system stems from the fact the two units are notphysically connected by wire, but are coupled inductively, i.e. by theuse of electromagnetic radiation, typically in the range from 5-1,000kHz, but also up to 1 GHz and higher.

FIG. 3 is a flow chart illustrating the communication between thetransponder 12 and a remote scanner read/write device 18. Thetransponder 12 associated with the solid phase beads 10 is encoded withdata sent by electromagnetic waves from a remote scanner read/writedevice 18. After the assay steps are completed, the beads 10 areanalyzed to detect the presence of a label indicative of binding ofanalyte and those that show the presence of the label are decoded. Thescanner 18 sends a signal to the transponder 12. In response to thesignal, the transponder 12 transmits the encoded data to the scanner 18.

Some transponders similar to those used in this invention are availablecommercially. Bio Medic Data Systems Inc. (BMDS, 255 West Spring ValleyAve., Maywood, N.J.) manufactures a programmable transponder for use inlaboratory animal identification. The transponder is implanted in thebody of an animal, such as a mouse, and is glass-encapsulated to protectthe electronics inside the transponder from the environment. One of thetransponders manufactured by this corporation, model# IPTT-100, hasdimensions of 14×2.2×2.2 mm and weighs 120 mg. The transponder isuser-programmable with up to 16 alphanumeric characters, the 16th letterprogrammable independently of the other 15 letters, and has a built-intemperature sensor as well. The electronic animal monitoring system(ELAMS) includes also a scanner read/write system to encode or read dataon/from the transponder. The construction of the transponder and scanneris described in U.S. Pat. Nos. 5,250,944, 5,252,962 and 5,262,772, thedisclosures of which are incorporated herein by reference. Other similartransponder-scanner systems include a multi-memory electronicidentification tag (U.S. Pat. No. 5,257,011) manufactured by AVIDCorporation (Norco, Calif.) and a system made by TEMIC-Telefunken(Eching, Germany). AVID's transponder has dimensions of 1 mm×1 mm×11 mm,and can encode 96 bits of information, programmed by the user. Thepresent invention can be practiced with different transponders, whichmight be of different dimensions and have different electronic memorycapacity.

The commercially available transponders are relatively large in size.The speed at which the transponders may be decoded is limited by thecarrier frequency and the method of transmitting the data. In typicalsignal transmission schemes, the data are encoded by modulating eitherthe amplitude, frequency or phase of the carrier. Depending on themodulation method chosen, compression schemes, transmission environment,noise and other factors, the rate of the signal transmission is withintwo orders of magnitude of the carrier frequency. For example, a carrierfrequency of 1,000 Hz corresponds to rates of 10 to 100,000 bits persecond (bps). At the rate 10,000 bps the transmission of 100 bits willtake 0.01 sec. The carrier frequency can be several orders of magnitudehigher than 1,000 Hz, so the transmission rates can be proportionallyhigher as well.

Therefore, the limiting factor in the screening process is the speed atwhich the transport mechanism carries the transponders through the readwindow of the fluorometer/scanner device. The rate of movement of smallparticles or cells is 10⁴ -10⁵ per second in state-of-the-art flowcytometers. A flow cytometer may be used to practice the presentinvention, if two conditions are met: (1) the transponders are smallenough to pass through the flow chamber, and (2) the design of the flowchamber of the flow cytometer is modified to include an antenna forcollecting the electromagnetic radiation emitted by transponders.

A miniature transponder is depicted in FIGS. 4 and 5. The source of theelectrical power for the transponder 12a is at least one photovoltaiccell 40 within the transponder 12a, illuminated by light, preferablyfrom a laser (not shown). The same light also induces the fluorescenceof the fluorogenic molecules immobilized on the surface of thetransponder 12a. The transponder 12a 9 includes a memory element 42 thatmay be of the EEPROM type. The contents of the memory is converted fromthe digital form to the analog form by a Digital-to-Analog converter 44mounted on the transponder 12a. The signal is amplified by an amplifier46, mixed with the carrier signal produced by an oscillator 48, andemitted to the outside of the transponder 12a by an antenna 50.

The contents of the transponder memory can be permanently encoded duringthe manufacturing process of the transponder, different batches oftransponders being differently encoded. Preferably, the memory of thetransponder is user-programmable, and is encoded by the user justbefore, during, or just after the biological material is deposited onthe surface of the transponder. A user-programmable transponder 12a musthave the "write" feature enabled by the antenna 50, amplifier 44 and theAnalog-to-Digital converter 46 manufactured on the transponder 12a, aswell as the dedicated scanner read/write device 27.

The advantages of the transponder of FIGS. 4 and 5 are several-fold.First, the dimension of the transponder is reduced, since most of thevolume of current transponders is occupied by the solenoid. The designdiscussed above will enable the production of cubic transponders on theorder of 0.01 to 1.0 mm along a side, preferably 0.05 to 0.2 mm.

Second, a large number of transponders can be manufactured on a singlesilicon wafer, and no further assembly would be required to attach thesolenoid to the VLSI chip. As depicted schematically in FIG. 5, asilicon wafer 60 is simply cut to yield active transponders 12a. Third,the transponder, according the new design, will not need the glasscapsule as an enclosure, further reducing the size of the transponder.Silicon dioxide (SiO₂) would constitute a significant portion of thesurface of the transponder, and SiO₂ has chemical properties which arevery similar to glass in terms of the feasibility of derivatization orimmobilization of biomolecules. Alternatively, microtransponders may becoated with a variety of materials, including plastic, latex and thelike.

Finally, most important, the narrow focus of the beam of the laser lightwould enable only one transponder to be active at a time, significantlyreducing the noise level. Advanced user-programmability is desirable aswell, various memory registers need to be addressable independently(writing in one register should not erase the contents of otherregisters).

FIG. 6 shows the analytical instrumentation and transport system used inan embodiment of the present invention. A quartz tube 20 is mounted inthe readout window 22 of a fluorometer 24. The quartz tube 20 isconnected to a metal funnel 26. The length of the quartz tube 20 issimilar to the dimensions of the transponder 12. Transponders 12 are fedinto the metal funnel 26, and pass from the funnel 26 into the quartztube 20, where the fluorescence is read by the fluorometer 24 and thetransponder 12 is decoded by the scanner 27, and then exit through ametal tube 28 and are conducted to a collection vessel (not shown). Themetal funnel 26 and metal tube 28 are made of metal shield transponders12 outside of the read window 22 by shielding from the electromagneticsignal from the scanner 27. This shielding prevents the scanner signalfrom reaching more than one transponder 12, causing multipletransponders 12 to be decoded.

Minimal modification of the fluorometer 24 would be needed in thevicinity of the location that the tube occupies at the readout moment toallow for positioning of the transponder reading device. To assurecompatibility with existing assays, the glass surrounding thetransponder could be coated with plastic currently used to manufacturebeads.

In a preferred design, depicted in FIG. 4, a metal coil antenna 30 iswrapped around the flow cell 32 of a flow cytometer 29. The transponders12 pass through the flow cell 32, and are decoded by the scanner device27. The signal carrying the data sent from the transponders 12 isamplified by a first amplifier 34 and processed by the scanning device27. As the transponders 12 are decoded, fluorescence from thetransponders 12 is detected and analyzed by the flow cytometer 29.

In one embodiment of the present invention, multiple passes of the sameset of transponders through the detector are implemented. Thetemperature is incrementally increased at each pass, the purpose beingto gradually increase the stringency of binding, and to reduce effectsof non-specific binding of the target to the probes on the transponders.After the detection step is completed, the transponders can bereconditioned for further use in another experiment by stripping thebiomolecular coating chemically, and clearing the memory. If the targetDNA is labeled with a fluorophore, an alternative to chemical strippingis the extensive wash at high temperature to dissociate and removenon-covalently bound target DNA.

Upon completion of the analysis step and decoding of the transponders,the data is analyzed to determine the sequence by correlating thesequence of the oligonucleotide probe immobilized on the transponder andthe fluorescence readout of the transponder carrying the probe. The typeof data related to the fluorescence readout may vary depending of theprocedures used. If the target DNA is labeled with the fluorophore priorto annealing, or if the label is a single fluorophore, introduced in achain extension reaction with a DNA polymerase after annealing, thefluorescence readout is simply the intensity of the fluorescence.Alternatively, if four ddNTPs are used with the purpose of identifyingthe nucleotide residue proximal to the 3' end of the probe or target DNA(depending on the design of the experiment), the fluorescence readout isboth the intensity and the information about the wavelength of maximalfluorescence. The form of the data set is analogous to that yielded bysequencing by hybridization methods(SBH), and algorithms previouslydeveloped for SBH can also be used for digital sequencing (Drmanac R. etal., 1993, DNA Sequence Determination By Hybridization: A Strategy ForEfficient Large-Scale Sequencing. Science 260, 1649-1652).

EXAMPLE 1 Immobilization Of DNA Probes On Transponders Using TheChemical Synthesis Of DNA

Nucleic acids can be covalently linked to glass by direct chemicalsynthesis on a glass support. To prepare the support, 5'-dimethoxytritylthymidine is reacted with one equivalent of tolylene-2,6-diisocyanate inthe presence of one equivalent of N-ethyldiisopropylamine as a catalystin pyridine/1,2-dichloroethane to generate the monoisocyanate. Themonoisocyanate is not isolated, but is added directly to the alkylamineglass support, i.e. the aminopropyltriethoxysilane-derivatized glasssurface of the transponders. The procedure is described in detail in B.S. Sproat and D. M. Brown, A New Linkage For Solid Phase Synthesis OfOligodeoxyribonucleotides, Nucleic Acids Res. 13, 2979-2987, 1985. Suchthymidine-derivatized support containing the stable nucleoside-urethanelinkage is directly used for the chemical synthesis ofoligodeoxynucleotides using a manual synthesis protocol on sinteredfunnels as described before (Caruthers, M. H. et al.,Deoxyoligonucleotide synthesis via the phosphoramidite method. In: GeneAmplification and Analysis, Vol III (T. S. Papas et al., Eds.).Elsevier/North Holland, Amsterdam), using standard phosphoramidite-basedDNA synthesis reagent. The thymidine-urethane linker is resistant tocleavage with base during the deprotection, and the resulting product isthe deprotected oligonucleotide attached to the glass surface of thetransponder through the urethane-thymidilate linker.

EXAMPLE 2 Determination Of The Sequence Of A Three Nucleotide RegionUsing AVID Transponders

The target DNA is a single-stranded 50-residue longoligodeoxynucleotide. It is obtained from a PCR reaction using a5'-fluoresceinated oligonucleotide as one of the primers, after linearlyamplifying DNA. DNA used in the experiment is purified after PCR. Mostof the sequence of 47 nucleotides in the 50 nt target is known, exceptfor a region consisting of three residues, residue numbers 18-20, markedas NNN in the figure below:

          transponder-linker-GGTACTGCXXXACCTTCCA-3'                                                                     (Seq ID No:1)                                                      .linevert split..linevert split..linevert                                                split..linevert split..linevert                                               split..linevert split..linevert                                               split..linevert split.   .linevert                                            split..linevert split..linevert                                               split..linevert split..linevert                                               split..linevert split..linevert                                               split..linevert split.                    3'-ACGTTAAGCCCAGTATGCCATACCATGACGNNNTGGAAGGTAGAGATACT-5'-fluor (Seq ID                                            No:2)                                        ↑         ↑         ↑         ↑                                                        ↑         ↑                      50        40       30        20        10         1                  

The symbol ".linevert split." denotes the complementarity of nucleotidesin the two sequences. There are exactly 64 possible sequences for5'-GGTACTGCXXXACCTTCCA (SEQ ID NO:1), where X can be any nucleotideresidue (A, C, G or T). 64 sets of AVID transponders are prepared, eachset having a different oligonucleotide probe (upper sequence in thefigure above) synthesized on the surface of the transponder usingstandard phosphoramidite chemistry as described in Example 1. The linkerhas the (dT)₁₀ sequence to facilitate annealing. The transponders areelectronically encoded with a number 1 through 64, the numbercorresponding to the sequence of the oligonucleotide bound to thetransponder. 64 transponders, each from a different set, are put into a10 ml vessel. A solution containing the target (5 ml volume) is thenadded to the vessel, the contents of the vessel is heated to 95° C.,cooled slowly over the period of 10 min to the experimentallypredetermined wash temperature T_(wash) (T_(wash) can range from roomtemperature to 60° C., data not shown). After the annealing step, thetransponders were extensively washed at temperature T_(wash), and thenstored in a buffer at room temperature.

Each of the 64 transponders was subjected to both fluorescencemeasurement and electronic decoding of the tag. As a result, each of 64possible sequences was associated with a number, fluorescence readout,that indicated whether the template had annealed to the oligonucleotideon the transponder, thus providing the information about the sequence ofthe 3 nt region.

EXAMPLE 3 Determination Of The Sequence Of A Two Nucleotide Region UsingCombinatorial Oligonucleotide Synthesis On BMDS Transponders

The target was a single-stranded 50-residue long oligonucleotide. It wasobtained from a PCR reaction using a 5'-fluoresceinated oligonucleotideprimer, after linearly amplifying DNA. DNA used in the experiment ispurified after PCR. Most of the sequence of 48 nucleotides in the 50 nttemplate was known, except for the region of two residues, residuenumbers 18-19, marked as NN in the figure below:

          transponder-linker-GGTACTGCAXXACCTTCCA-3'                                                                     (Seq ID No:3)                                                      .linevert split..linevert split..linevert                                                split..linevert split..linevert                                               split..linevert split..linevert                                               split..linevert split..linevert                                               split.  .linevert split..linevert                                             split..linevert split..linevert                                               split..linevert split..linevert                                               split..linevert split..linevert                                               split.                                    3'-ACGTTAAGCCCAGTATGCCATACCATGACGTNNTGGAAGGTAGAGATACT-5'-fluor (Seq ID                                            No:4)                                        ↑         ↑         ↑         ↑                                                        ↑         ↑                                                           50        40       30        20                                                  10         1                 

The symbol ".linevert split." denotes the complementarity of nucleotidesin the two sequences. The linker has the (dT)₁₀ sequence to facilitateannealing. There are exactly 16 possible sequences of oligonucleotides5'-GGTACTGCAXXACCTTCCA (SEQ ID NO:3), where X can be any nucleotideresidue (A, C, G or T). These sequences are synthesized in acombinatorial fashion on 200 BMDS transponders (14×2×2 mm, volume ofeach transponder is about 50 μl) as follows. First, the underivatizedglass surface of the transponders is treated as described in Example 1to introduce the functional hydroxy groups on the surface oftransponders. Then, the transponders are subjected to 18 rounds of solidphase oligonucleotide synthesis using standard phosphoramidite chemistryto sequentially synthesize the following residues:TTTTTTTTTTGGTACTGCA-3(SEQ ID NO:5). The transponders are split into fourgroups of approximately equal numbers (i.e. 50). Each of the four groupsis subjected to a single cycle of solid phase oligonucleotide synthesiswith a different nucleotide phosphoramidite. That is, transponders fromgroup 1 are derivatized with adenosine phosphoramidite (A), group2--cytosine phosphoramidite (C), group 3--guanosine phosphoramidite (G),and group 4--thymidine phosphoramidite (T). The volume of each of thefour condensations is about 4 ml. After completing the first cycle, the4 groups are subjected to electronic encoding with the residueabbreviations given above. Thus, the first alphanumeric characterindicates which condensation takes place in the first cycle ofcombinatorial synthesis. Then, all the transponders are pooled togetherand mixed thoroughly, and then split into new four groups havingapproximately the same number of transponders. These four new groups aresubjected to the second combinatorial coupling step. The scheme for thesecond coupling is identical to that of the first coupling. Electronicencoding after the second cycle adds a second alphanumeric character,corresponding to the nucleoside coupled in the second synthesis cycle.As a result, each transponder is encoded with two alphanumericcharacters which identify the sequence of two nucleotide residues at the3' end of the oligonucleotide attached to the surface of thetransponder. The transponders having oligonucleotides coupled to theirsurfaces are immersed in a 20 ml vessel in the solution containing thetemplate, the contents of the vessel is heated to 95° C., cooled slowlyover the period of 10 min to the experimentally predetermined annealingtemperature T_(ann) (T_(ann) can range from room temperature to 60° C.,data not shown). After the annealing step, the transponders areextensively washed at temperature T_(ann). The fluorescence ofindividual transponders is recorded and the transponder memories aredecoded. Strong fluorescence indicates that the complement of thedinucleotide sequence bound to the transponder is present in the target.

EXAMPLE 4

Determination Of The Sequence Of A Three Nucleotide Region Using ASingle-Nucleotide Extension By DNA Polymerase

The target was a single-stranded 50-residue long oligonucleotideobtained from a PCR reaction after linearly amplifying DNA. The targetDNA does not carry any fluorophore label. Most of the sequence of 47nucleotides in the 50 nt target is known, except for the region of threeresidues, residue numbers 18-20, marked as NNN in the figure below:

                                  5'            3'                                  transponder linker-GTATGGTACTGCAA        oligo 9 (Seq ID NO:6)                 -                ...-GTATGGTACTGCAC              8 (Seq ID NO:7)                                               -                ...-GTATGGTACTGCAG                                                    7 (Seq ID NO:8)                     -                ...-GTATGGTACTGCAT              6 (Seq ID NO:9)                                               -                ...-GGTATGGTACTGCA                                                    5 (Seq ID NO:10)                    -                ...-GGTATGGTACTGCC              4 (Seq ID NO:11)                                              -                ...-GGTATGGTACTGCG                                                    3 (Seq ID NO:12)                    -                ...-GGTATGGTACTGCT              2 (Seq ID NO:13)                                              -                ...-CGGTATGGTACTGC                                                    1 (Seq ID NO:14)                                      .linevert split..linevert split..linevert split..line                                      vert split..linevert split..linevert                                          split..linevert split..linevert                                               split..linevert split..linevert                                               split..linevert split..linevert                                               split..linevert split..linevert split.                                         3'-ACGTTAAGCCCAGTATGCCATACCATGACGNNNTGG                                      AAGGTAGAGATACT-5' (Seq ID NO:2)                  50        40        30        20        10       1                   

The symbol ".linevert split." denotes the complementarity of nucleotidesin the two sequences. The linker has the (dT)₁₀ sequence to facilitateannealing. Nine groups of five AVID transponders are derivatized by thechemical synthesis of oligonucleotides 1 through 9 as shown in thefigure above, and electronically encoded with numbers 1 through 9,respectively. The transponders are placed in one 5 ml vessel, and 2 mlsof the solution containing the target (final concentration 10 nM to 10μM) is added to the vessel. The contents of the vessel are heated to 95°C., cooled slowly over the period of 10 min to the experimentallypredetermined extension temperature T_(ex) (T_(ex) can range from roomtemperature to 60° C., data not shown). After the annealing step, thetransponders are extensively washed at temperature T_(ex). DNApolymerase and four dideoxynucleotide triphosphates, ddATP, ddCTP, ddGTPand ddTTP (obtained from Applied Biosystems) derivatized with fourdifferent fluorophores, such as those used in Applied Biosystemsautomated sequencers are then added to the vessel, and the vessel isincubated at T_(ex) for 15 minutes. Subsequently, the transponders areextensively washed at the wash temperature T_(wash), A set of datapoints, each in the form of (oligonucleotide₁₃ sequence, fluorescence₋₋intensity, wavelength₋₋ of₋₋ maximum₋₋ fluorescence), is obtained. Forhighly fluorescent transponders, the wavelength of maximum fluorescenceindicates the residue type, and the number encoded on the transponderindicates the position of the residue in the chain, thus defining thesequence of the 3 nt region of the target.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 14                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - GGTACTGCNN NACCTTCCA             - #                  - #                      - # 19                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - TCATAGAGAT GGAAGGTNNN GCAGTACCAT ACCGTATGAC CCGAATTGC  - #                   50                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - GGTACTGCAN NACCTTCCA             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - TCATAGAGAT GGAAGGTNNT GCAGTACCAT ACCGTATGAC CCGAATTGC  - #                   50                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -  - -     (ii) MOLECULE TYPE: DNA (genomic)                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - TTTTTTTTTT GGTACTGCA             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - GTATGGTACT GCAA              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - GTATGGTACT GCAC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - GTATGGTACT GCAG              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - GTATGGTACT GCAT              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - GGTATGGTAC TGCA              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - GGTATGGTAC TGCC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - GGTATGGTAC TGCG              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - GGTATGGTAC TGCT              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - CGGTATGGTA CTGC              - #                  - #                      - #     14                                                                 __________________________________________________________________________

I claim:
 1. A solid phase support comprising:(a) a transponder which isa monolithic integrated circuit which can transmit via electromagneticradiation data stored within said integrated circuit; and (b) anoligonucleotide probe attached to the surface of the integrated circuit,wherein said probe is capable of binding to a nucleic acid.
 2. A solidphase support comprising:(a) a transponder which is a monolithicintegrated circuit which can transmit via electromagnetic radiation datastored within said integrated circuit; (b) at least one layer of acoating deposited on the surface of the integrated circuit, (c) anoligonucleotide probe attached to the coating layer, wherein said probeis capable of binding to a target nucleic acid.
 3. The solid phasesupport of claim 1, wherein said monolithic integrated circuit comprisesa photovoltaic cell that can be activated by light.
 4. The solid phasesupport of claim 3, wherein said data stored in the memory of saidmonolithic integrated circuit are encoded by a manufacturer of saidintegrated circuit.
 5. The solid phase support of claim 4, wherein saiddata stored in the memory of the monolithic integrated circuit areencoded by a user with a scanner device.
 6. The solid phase support ofclaim 2, wherein said monolithic integrated circuit comprises (a) aphotovoltaic cell that can be activated by light and (b) said coating isat least partially transmissible to light.
 7. The solid phase support ofclaim 6, wherein said data stored in the memory of said monolithicintegrated circuit are encoded by a manufacturer of said integratedcircuit.
 8. The solid phase support of claim 7, wherein said data storedin said memory of the monolithic integrated circuit are encoded by auser with a scanner device.
 9. A kit for determining the sequence oftarget nucleic acids in a sample, comprising:(a) at least one assayvessel, containing at least one solid phase particle having (i) atransponder and (ii) an oligonucleotide probe bound to its surface; and(b) at least one label reagent.
 10. The kit of claim 9, wherein thelabel reagent comprises a reagent that labels the target nucleic acid.11. The kit of claim 9, wherein the label reagent comprises a labelednucleotide.
 12. The kit of claim 9, further comprising:(a) a samplediluent buffer solution; and (b) an enzyme reaction buffer solution. 13.A kit of claim 9, wherein the transponder is a monolithic integratedcircuit.
 14. The kit of claim 13, wherein the label reagent comprises areagent that labels the target nucleic acid.
 15. The kit of claim 13,wherein the label reagent comprises a labeled nucleotide for use in achain extension reaction using DNA polymerase.
 16. The kit of claim 13,further comprising:(a) a sample diluent buffer solution; and (b) anenzyme reaction buffer solution.